Catalog 2016

Catalog 2016 Exosome experts since 2007

Optimized solutions for Exosome Research

Lyophilized Exosome Standards• Exosome Isolation Tools • Exosome Quantification Kits • RNA Extraction Kits • DNA Extraction kit • Exosome binding Antibodies • Services

HansaBioMed LLC HansaBioMed LLC is a Company entirely dedicated to research and development of products in the field of Exosome Sciences.

Our facilities are located in Tallinn (Estonia), in the Tallinn Technology Park (Tehnopol), and in Italy at Exosomics Siena (Siena).

HansaBioMed offers the widest choice and selection of products dedicated to exosome research for Life Science applications available on the global market today.

We are the oldest and only company focused specificly on Exosome isolation and analysis, producing kits that are easy to use and high quality. Some of the products available are Exosome Standards, Exosome binding Antibodies and kits for enrichment of specific exosome subpopulations.

The full range of our products and services is described on this catalogue and can be purchased directly from our on-line shop: (www.exotest.eu/online_orders/ ).

1

Catalog 2016: Table of Contents Section 1: Lyophilized Exosome Standards • •

3

Lyophilized Exosome Standards from human biofluids Lyophilized Exosome Standards from cell culture media

Section 2: Exosome Isolation Tools • • •

15

EXO-Prep Immunoplates Immunobeads

Section 3: Exosome Quantification kits • •

29

ExoTEST Ready to use kits Exo-FACS Ready to use kits TM

Section 4: Exosome RNA Extraction kits • • •

37

EXO-TotalRNA TumorEXO-TotalRNA RNA basic kit

Section 5: Exosome DNA Extraction kits • •

43

EXO-DNA/PS: Circulating and exosome-associated DNA from plasma and serum EXO-DNA/UC: Circulating and exosome-associated DNA from urine and cell media

Section 6: Antibodies for Exosome Research • •

HBM-Monoclonal antibodies HBM-Polyclonal antibodies

Section 7: Custom Services and Collaborations • • • • •

47

Exosome and microvesicles purification and analysis RNA and DNA profiling and Next-Gen-Sequencing (NGS) Custom products Additional services and project collaborations Cell fractionation and cell proliferation kits 2016

2

59

1

Lyophilized Exosome Standards

Summary Section 1 Introduction

4

Lyophilized Exosome Standards from body fluids or cell culture media

5

Performances

7

Exosome Standards and cell lysates on request

9

1

Lyophilized Exosome Standards Introduction

Applications: •

Assay calibration

•

Control (spike-in) for exosome quantification

•

Protein marker analysis using different techniques

•

Extraction and analysis of exosome nucleic acid

•

Standardized positive controls for immunocapture performance evaluation

•

Flow cytometry

•

Electron microscopy

Advantages •

Highly pure

•

Easy to reconstitute

•

Easy to ship and store (+4°C)

•

Long term storage stability (36 months)

•

Better performances then competitors

•

Exosomes available from a large cell line bank (over 100 cell lines)

•

Exosomes availabile from different body fluids (Plasma, Serum, Urine, Saliva)

Exosomes are small endosome derived lipid nanoparticles (50-120 nm) actively secreted by exocytosis by most living cells. Exosome release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both quantity and molecular composition of released exosomes depend on the physiological state of the parental cells.

Lyophilization is the ideal technique for preserving the long-term stability of exosomes at 4ºC. Lyophilized exosomes can be used as control standards for multiple applications including FACS, WB, ELISA and as calibration standards for quantitation of exosome-derived markers from biological samples.

Lyophilized exosomes are easy to ship and store, and are stable for over 36 months at 4°C. Purified and lyophilized exosomes are obtained from a variety of biological sources: cell culture supernatant, human plasma, serum, urine and saliva. Exosomes are purified following a combination of ultracentrifugation and microfiltration steps.

Exosomes are subsequently quantified and validated for overall protein content and particle number by NTA (Nanoparticles Tracking Analysis) with NanoSight LM10.

Lyophilization does not affect the stability of purified exosomes and the expression levels of their exosome markers (proteins and nucleic acids).

1-Exosome Standard from Human Body Fluids

Purified exosomes from human Plasma, Serum and Urine (pools of healthy donors)

2-Exosome Standard from Cell Culture Supernatants

Purified exosomes from supernatants of a wide list of human tumor cell lines

3-Cell lysates

Whole cell lysates from a wide list of human tumor cell lines

4

Lyophilized Exosome Standards Exosome Standards from Human Body Fluids and Cell Media HansaBioMed provides lyophilized Exosome Standards from Human Plasma, Serum, Urine and Saliva pool of certificated healthy donors and from different cell lines in continous culture. All Exosome Standards are quantified and validated for overall protein content and particle number by NTA with NanoSight LM10. They can be easily reconstituted by adding the appropriate volume of deionized water (MilliQ) and, once reconstitued, they can be stored at -20°C for up to 1 month and at - 80°C for up to 6 months.

Cat Code

Contents

Package size (#)

Lyophilized Exosome Standards from Human Biofluids Lyophilized Exosome from Human Plasma of healthy donors HBM-PEP100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-PEP30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosome from Human Serum of healthy donors HBM-PES100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-PES30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

1 Storage of Lyophilized Exosome Standards.

Lyophilized Exosome Standards can be stored at 4°C for up to 36 months.

Storage of reconstituded exosomes.

After reconstitution Exosomes can be stored at -20°C for up to one month or at -80°C for up to six months. Avoid freeze and thaw cycles.

Lyophilized Exosome from Human Urine of healthy donors HBM-PEU100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-PEP30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosome from Human Saliva of healthy donors HBM-PESL100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-PESL30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes Standards from Cell Culture Media

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/

Lyophilized Exosomes from COLO1 cell line (Human colon carcinoma) HBM-COLO-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-COLO-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from MM1 cell line (Human melanoma) HBM-MM1-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-MM1-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from BLCL21 cell line (EBV transformed lymphoblastoid B cells) HBM-BLCL-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-BLCL-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from U87 MG cell line (Human glioblastoma astrocytoma) HBM-U87-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-U87-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from SK-N-SH cell line (Human neuroblastoma) HBM-SK-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-SK-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from HCT116 cell line (Human colon carcinoma) HBM-HCT-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-HCT-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

5

US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

1 Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Lyophilized Exosome Standards

Cat Code

Contents

Package size (#)

Lyophilized Exosomes Standards from Cell Culture Media Lyophilized Exosomes from PC3 cell line (Human Prostate adenocarcinoma grade IV) HBM-PC3-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-PC3-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from BPH-1 cell line (Human being prostatic hyperplasia) HBM-BPH-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-BPH-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from DAUDI cell line (Human Burkitt Lymphoma) HBM-DAUDI-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-DAUDI-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from A549 cell line (Lung carcinoma)

Info and Technical support For informations and techincal support contact us at: info@hansabiomed.eu

HBM-A549-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-A549-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from K-562 cell line (Pleural effusion, leukemia chronic myelogenous) HBM-K562-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-K562-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

Lyophilized Exosomes from B16F10 mouse cell line (mouse melanoma cell line) HBM-B16-100/#

100 μg (>1x10^10 particles)

2 vials

4 vials

6 vials

HBM-B16-30/#

30 μg (>1x10^8 particles)

2 vials

4 vials

6 vials

HBM can provide, upon request, Exosome Standards and Cell Lysates from more than 100 different tumor cell lines.

Cell Lines informations All HansaBioMed Exosome Standards from Cell Lines are produced using Cell Lines from the Cell Bank of the Interlab Cell Line Collection of the IRCCS AUO S.Martino IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. To order Cell Lines refer directly to:

References Sitar, Simona, et al. “Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation.” Analytical chemistry (2015).

Gardiner, Chris, et al. “Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles.” Journal of extracellular vesicles 3 (2014).

Ferrante, Sarah C., et al. “Adipocyte-derived exosomal miRNAs: a novel mechanism for obesity-related disease.” Pediatric research 77.3 (2014): 447-454.

www.iclc.it/indexpi.html.

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1

Lyophilized Exosome Standards Performances Lyophilization is the ideal method for preserving exosome stability.

Expression of exosomal markers was assayed with different techniques (WB, FACS, ELISA) and nanovesicle count was measured with NTA. Lyophilization did not substantially affect exosome count or biomarker expression compared to other storage methods (Fig 1, 2, 3, 4).Comparing different storage methods of exosome standards (fresh vs. frozen vs lyophilized) with an anti-CD81 ELISA assay, the loss of signal compared to fresh material is minimal when using lyophilized exosomes (CV - 15%) (Fig 4).

Fresh Exosomes

Lyophilized Exosomes

2. Comparison of exosomal markers on fresh and lyophilized exosomes.

1. Western Blot comparison of exosomal markers on fresh (F), frozen (-20ºC) and lyophilized exosomes (L)

1

3. Comparative Nanosight analysis of freshly purified (lower panel) and lyophilized plasma exosomes (upper panel).

2

3

4. ExoTEST™ comparative detection of CD81 on HMBMM1 derived exosomes: 1- Fresh exosomes; 2- Frozen exosomes (-20°C); 3- Lyophilized exosomes

HBM Exosome Standards are the ideal tool for your exosome studies.

HBM Exosome Standars are suitable for many different applications and techniques. Examples reported in figures show a profile of common exosomal markers in human plasma exosomes (HBM-PEP, Fig 5) and Beta-Actin transcript amplified from RNA obtained from COLO-100, MM1-100 and BLCL21-100 standards (Fig 6) 1 0,9 0,8

O.D. 450 nm

0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 CD9

CD63

CD81

TSTA3

TM9SF4

HSP70

blank

5. Profile of common exosomal markers in plasma exosome standard. HBM-PEP-30 is used to quantify each tested marker

6. β-Actin transcript amplification from total-RNA extracted from HBM-Exosome Standard COLO-100, MM1100, BLCL-100

7

1

Lyophilized Exosome Standards

HBM Exosome Standards guarantee higher purity and better performances over competitors.

HBM Exosome Standard from human serum (HBM-PES) were compared to human serum exosome standard from a Competitor. Exosomal markers CD9 and CD63 from 50 μg of both serum exosome standards were detected using an ELISA assay (fig 6). HBM Standard (50 μg) generated the highest signal, whereas the signal from Competitor Standard (50 μg) was lower than 30 μg of HBM Standard. In a second test, 50 μg of HBM and Competitor’s Standards were serially diluted to design a standard curve for exosome quantification (fig 7). The standard curve generated with HBM Standard was linear (R2 = 0,9876) within the concentration range and suitable for exosome quantification.

1,8

CD9 detection

1,6

1,2 R² = 0,9876

1

1,2

HBM-PES-50 ug

O.D.450 nm

O.D 450 nm

1,4

1 HBM-PES-30 ug

0,8 0,6

Competitor Serum exo 50 ug

0,4

0,8 0,6

HBM-PES-50 ug

0,4

Competitor Serum exo 50 ug

0,2

0,2

0 0

0 CD9

CD63

10

20

30

Exosomes (ug)

Blank

6. ELISA quantification of HBM Exosome Standard vs Competitor’s standard (human serum) for exosomal markers CD9 and CD63

7. Standard curve for exosome quantification: HBM Exosome Standard vs. Competitor’s standard

Why to choose HBM Exosome Standards Characteristics Amount per vial

HansaBioMed Exosome Standard 100 μg

50 μg

Ultracentrifuge

Precipitation Reagent

Nanoparticles/ml (average)

> 1x10^10

>1x10^6

Nanoparticles/ml in human serum exosomes (50 μg)

3,75x10^10 p/ml

2,29x10^9 p/ml

Lyophilized

Frozen

4°C (lyophilized)

-20°C

Method of isolation

Final form Storage temperature

HBM Exosome Standards are purified using a combination of ultracentrifugation and microfiltration steps.

This is a tedious and time-consuming methodology but it guarantees high purity of exosomes, with low contamination from other microvesicles, previously eliminated through microfiltration and differential centrifugation cycles. Conversely, chemical reagents currently used for vesicle isolation precipitate both exosomes and larger vesicles, protein-protein and protein-RNA complexes, regardless of their origin. This impairs the purity of the sample. Lyophilization also allows easy and longer term storage of purified exosomes.

Competitor’s Exosome Standards

Expire time

36 months

24 months

Price (1 vial)

125 € x 100 ug

over 300€ x 50 ug

8

Lyophilized Exosome Standards Exosome Standards and Cell lysates on request HBM can provide Purified Exosome Standard or whole Cell Lysates from the cell lines in following list.

List of available Cell Lines for Exosome Standards purification or Cell Lysates Cell Line Name

Tissue

Tumor/Pathology Description

peripheral blood

leukemia, pre-B cell

1301

blood

leukemia, acute lymphoblastic, T cell

5637

bladder

carcinoma

380

8305C

thyroid

carcinoma, undifferentiated

A 2058

skin

melanoma, metastatic

A-172

-

glioblastoma

A-204

muscle

rhabdomyosarcoma

A2780

ovary

adenocarcinoma

A-498

kidney

adenocarcinoma

A-704

kidney

adenocarcinoma

ACHN

kidney

adenocarcinoma

-

neuroblastoma

BICR 18

larynx

squamous cell carcinoma

BT-474

breast

carcinoma, ductal

ACN

BT-549

breast

carcinoma, ductal

BV-173*

peripheral blood

leukemia, pre-B cell

BxPC-3

pancreas

adenocarcinoma

C33A

cervix

carcinoma

CA46

ascitic fluid

lymphoma, Burkitt

Caco-2

colon

adenocarcinoma

Caki-2

kidney

carcinoma

Calu-1

lung

carcinoma, epidermoid, grade III

CaSki

cervix

cervix carcinoma, epidermoid

CFPAC-1

pancreas

adenocarcinoma

CM-S/Tum

bone marrow

monocyte tumor

CM-S/un

bone marrow

monocyte tumor

colon

colorectal adenocarcinoma

COLO 205 COLO 320DMF

colon

adenocarcinoma

lung, derived from pleural fluid

adenocarcinoma

COLO 741

colon

carcinoma, pelvic wall metastasis

COLO 800

subcutaneous nodule

melanoma

COLO 853

lymph node

melanoma

COLO 858

lymph node

melanoma

lung

carcinoma, large cell

COLO 699N

COR-L23

9

1 Cell Lines informations All HansaBioMed Exosome Standards from Cell Lines are produced using Cell Lines from the Cell Bank of the Interlab Cell Line Collection of the IRCCS AUO S.Martino IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.

All cell lines are also available in vital form either frozen or in culture at the cell bank of the IRCCS AOU San Martino, Genova. To order Cell Lines refer directly to: www.iclc.it/indexpi.html.

Ordering Informations Exosome Standards and Cell Lysates from the available cell lines can be ordered by e-mail at the address: orders@hansabiomed.eu

Exosome Standards Exosome Standards from cell lines on request are available only as 100 Îźg size, 4 or 6 vials Catalog Code HBM-PEC-##-100/4(6) #- Name of cell line

* Epstein-Barr virus ** Transformed by Epstein-Barr virus *** type C, type B viruses

1

Lyophilized Exosome Standards

Cell Lysates Cell Lysates from the cell line list are available in package size of 1 or 2 vials, 100 μg each one.

Catalog Code: HBM- LYS-#-1(2) #- Name of cell line

Storage of Lyophilized Exosome Standards.

Lyophilized Exosome Standards can be stored at 4°C for up to 36 months.

Storage of reconstituted exosomes.

After reconstitution Exosomes can be stored at -20°C for up to one month or at -80°C for up to six months. Avoid freeze and thaw cycles.

* Epstein-Barr virus ** Transformed by Epstein-Barr virus *** type C, type B viruses

List of available Cell Lines for Exosome Standards purification or Cell Lysates Cell Line Name

Tissue

Tumor/Pathology Description

DBTRG.05MG

brain

glioblastoma

DLD-1

colon

adenocarcinoma

lung, pleural effusion

carcinoma, small cell

-

lymphoma, follicular, B cell

prostate

carcinoma

DMS-79 DOHH2* DU-145 FTC-133

thyroid

carcinoma, follicular

FTC-238

thyroid

carcinoma, follicular

G-361

skin

melanoma

GDM-1

peripheral blood

leukemia, acute myelomonocytic

GF-D8

peripheral blood

leukemia, acute myeloid

lymphocyte

lymphoma

HCT-15

colon

colorectal adenocarcinoma

HCT-8

intestine, ileocecal

ileocecal adenocarcinoma

HECV

umbilical cord

-

-

erythroleukemia

H9

HEL 92.1.7 HeLa

cervix

carcinoma, epitheloid

HeLa S3

cervix

carcinoma, epitheloid

-

chondrosarcoma

Hep G2

liver

hepatocellular carcinoma

HFFF2

foreskin, fetal

fibroblast, fetal

H-EMC-SS

HGC-27

stomach

carcinoma, undifferentiated

peripheral blood

leukemia, promyelocytic

HOS

bone

osteosarcoma

Hs578T

breast

carcinoma

Hs913T

derived from metastasis to lung

fibrosarcoma

HT 1197

bladder

carcinoma

HT-1080

HL-60

acetabulum

fibrosarcoma

HT-29

colon

adenocarcinoma, grade II

HuP-T3

pancreas

adenocarcinoma, ascitic fluid

HuP-T4

pancreas

adenocarcinoma, ascitic fluid

Hs913T

derived from metastasis to lung

fibrosarcoma

HT 1197

bladder

carcinoma

HT-1080

acetabulum

fibrosarcoma

HT-29

colon

adenocarcinoma, grade II

HuP-T3

pancreas

adenocarcinoma, ascitic fluid

HuP-T4

pancreas

adenocarcinoma, ascitic fluid

HUVEC

endothelium

umbelical vein endothelial cells

IMR-32

-

neuroblastoma

IMR-5

-

neuroblastoma

IST-MEL1

skin

melanoma

IST-MEL2

skin

melanoma

IST-MEL3

skin

melanoma

subcutaneous metastasis

melanoma, metastatic

IST-MES1

pleural effusion

mesothelioma

IST-MES2

pleural effusion

mesothelioma

IST-MELA 16

10

Lyophilized Exosome Standards

1

List of available Cell Lines for Exosome Standards purification or Cell Lysates Cell Line Name IST-SL1

Tissue

Tumor/Pathology Description

lung

carcinoma, small cell

-

lymphoma, follicular, B cell

oesophagus

carcinoma,squamous cell

LB4**

lymphocyte, B

paroxysmal nocturnal hemoglobinuria

LB-B7**

lymphocyte, B

paroxysmal nocturnal hemoglobinuria

LB-F9**

lymphocyte, B

paroxysmal nocturnal hemoglobinuria

prostate

adenocarcinoma

KARPAS-422* KYSE-30

LNCap.FGC LoVo

colon

adenocarcinoma

LS 180

colon

colorectal adenocarcinoma

M07e

peripheral blood

leukemia, acute megakaryoblastic

MCF7***

breast

adenocarcinoma

MDA-MB-415

breast

adenocarcinoma

MDA-MB-435S

breast

carcinoma, ductal

MDA-MB-436

mammary gland

adenocarcinoma

MDA-MB-453

breast

adenocarcinoma

MDA-MB-468

breast

adenocarcinoma

MeCo 05

skin

melanoma

MEG-01

bone marrow

leukemia, megakaryoblastic

MEGR 07

metastatic cutaneous nodule

melanoma

MeMo 05

lymph node, metastasis

melanoma

MEMOR 06

subcutaneous metastasis

malignant melanoma

MES-SA

uterus

sarcoma

MEWO

-

malignant melanoma

MG-63

-

osteosarcoma

MOLT-4

peripheral blood

leukemia, T cell

MONO-MAC-6

peripheral blood

leukemia,acute monocytic

MPP 89

pleural effusion

mesothelioma

MRC-5

lung, fetal

-

MSTO-211H

-

mesothelioma

NCI-H1650

lung

adenocarcinoma, bronchioalveolar carcnoma (smoker patient)

NCI-H1975

lung

adenocarcinoma, non-small cell (non-smoker patient)

NCI-H292

lung

carcinoma, mucoepidermoid

NCI-H727

lung

carcinoma, non-small cell

NT2-D1

testis

carcinoma, embryonal pluripotent

peripheral blood

leukemia, acute myeloid

MPP 89

pleural effusion

mesothelioma

MRC-5

lung, fetal

-

OCI-AML2

MSTO-211H

-

mesothelioma

NCI-H1650

lung

adenocarcinoma, bronchioalveolar carcnoma (smoker patient)

NCI-H1975

lung

adenocarcinoma, non-small cell (non-smoker patient)

NCI-H292

lung

carcinoma, mucoepidermoid

NCI-H727

lung

carcinoma, non-small cell

11

* Epstein-Barr virus ** Transformed by Epstein-Barr virus *** type C, type B viruses

1

Lyophilized Exosome Standards List of available Cell Lines for Exosome Standards purification or Cell Lysates Cell Line Name NT2-D1 OCI-AML2

testis

carcinoma, embryonal pluripotent leukemia, acute myeloid

ovary

teratocarcinoma

pleural effusion

leukemia, T cell

PSN1

pancreas

adenocarcinoma

RAJI

-

lymphoma, Burkitt

Rj2.2.5

-

lymphoma, Burkitt

thyroid

carcinoma, follicular

bone marrow

lymphoblastoid, EBV transformed

skin

melanoma

RO82-W-1 ROV-S** RPMI 7932 Saos-2

bone

osteosarcoma

bone marrow metastasis

neuroblastoma

SiHa

cervix

carcinoma, squamous cell

SK-BR-3

breast

adenocarcinoma

SH-SY5Y

SK-HEP-1

liver

adenocarcinoma

SK-LU-1

lung

adenocarcinoma, grade III, poorly differentiated

SK-MEL-24

skin

melanoma

SK-MEL-28

skin

melanoma

SK-MEL-5

skin

melanoma

SK-MES-1

lung

carcinoma, squamous cell

SK-N-AS SK-N-BE(2) SK-N-BE(2)-C

-

neuroblastoma

bone marrow

neuroblastoma

bone marrow

neuroblastoma

SK-N-F1

bone marrow metastasis

neuroblastoma

SUP-T1

pleural effusion

lymphoma, lymphoblastic, T cell

SW1353

bone

chondrosarcoma

SW48

colon

adenocarcinoma, grade IV

SW480

colon

adenocarcinoma, grade III-IV

SW620

colon

adenocarcinoma, metastasis to lymph node

SW837

rectum

adenocarcinoma, grade IV

T47D

breast

carcinoma, ductal

T84

colon

carcinoma, metastasis to lung

-

rhabdomyosarcoma

TE671 Subline 2 TF-1

* Epstein-Barr virus ** Transformed by Epstein-Barr virus *** type C, type B viruses

Tumor/Pathology Description

peripheral blood

PA-1 PF-382

Tissue

bone marrow

erythroleukemia

THP-1

peripheral blood

leukemia, acute monocytic

THP-1h

peripheral blood

leukemia, acute monocytic

THP-1l

peripheral blood

leukemia, acute monocytic

U251 MG

-

glioblastoma-astrocytoma, grade III

U87 MG

brain

glioblastoma-astrocytoma

U87/DK

brain

glioblastoma, transfected with binase (ATP binding site 721), mutated de 2-7 EGFR

U87/WT

brain

glioblastoma, transfected with EGFR

pleural effusion

lymphoma, histiocytic

U-937

12

Lyophilized Exosome Standards List of available Cell Lines for Exosome Standards purification or Cell Lysates Cell Line Name VA-ES-BJ

Tissue

Tumor/Pathology Description

skin

sarcoma, epitheloid

WiDr

colon

colorectal adenocarcinoma

Y79

-

retinoblastoma

13

1

1

Lyophilized Exosome Standards

14

2

Exosome Isolation Tools

Summary Section 2 Introduction

16

EXO-Prep

17

EXO-Prep performances

18

Immunoplates for Exosome capture and isolation

20

Immunoplate performances

22

Latex immunobeads for Exosome capture and isolation

24

Latex immunobead performances

26

2

Exosome Isolation Tools Introduction

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Ultracentrifugation is currently the gold standard methodology for exosomes isolation from biological fluids or cell supernatants. However it does not effectively isolate exosomes from small volume samples, it requires expensive equipment and it is time-consuming. To address these issues, the HBM team has developed and optimized several tools for isolating overall or specific exosome populations, in an efficient, faster and cheaper way. We present three classes of products:

Technical support For informations and techincal support contact us at: info@hansabiomed.eu

1-EXO-Prep

New and efficient one-step method for total exosome isolation from biofluids (i.e. plasma, serum and urine) and from cell culture media.

2-Immunoplates

For exosome immunocapture using generic or specific exosome-associated biomarkers (i.e. tumoral, neural and glial derived).

3-Immunobeads

Latex immunobeads for generic and specific exosome immunocapture from human biofluids or cell supernatants. Immunocaptured exosome can be recovered and used for downstream analyses.

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Exosome Isolation Tools EXO-Prep

Applications

EXO-Prep is a fast and convenient method of exosome isolation from biofluids and cell culture supernatants. Isolation with EXO-Prep is based on chemical precipitation. Samples are incubated with EXO-Prep solution on ice, so that exosomes will precipitate following centrifugation. The obtained pellet can be resuspended in PBS 1X or deionized water. The protocol is user friendly, timesaving (around 1 hour), and does not require capital laboratory equipment.

Isolated exosomes are suitable for a wide range of analyses, such as NTA, protein profiling by using different techniques (western blotting, ELISA, FACS), nucleic acid extraction and profiling of mRNA or small RNA markers.

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Single step isolation of exosomes from biofluids and cell supernatants.

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Isolate the overall exosome population in a sample.

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Isolated exosomes can be used for NTA analysis.

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Isolated exosomes are suitable for protein profiling (WB, ELISA, FACS).

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Isolated exosomes are suitable for nucleic acid extraction and profiling.

Advantages

Cat. Code

Volume

Reactions

EXO-Prep for Exosome Isolation from Plasma and Serum HBM-EXP-B5

5 ml

180 reactions Plasma, 80 reactions Serum

HBM-EXP-B10

10 ml

350 reactions Plasma, 160 reactions Serum

HBM-EXP-B20

20 ml

700 reactions Plasma, 320 reactions Serum

EXO-Prep for Exosome Isolation from Cell Media HBM-EXP-C25

25 ml

25 reactions

HBM-EXP-C50

50 ml

50 reactions

HBM-EXP-U25

30 ml

25 reactions

HBM-EXP-U50

60 ml

50 reactions

EXO-Prep for Exosome Isolation from Urine

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Time and money saving.

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No ultracentrifugation required.

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Easy and fast protocol.

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Isolate exosome from small volumes of sample (as low as 100 μl of plasma).

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Easy to store and ship (4°C).

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Exosome Isolation Tools Performances

EXO-Prep isolates exosomes in one single step from a small volume of sample NTA analysis (Fig 1) of plasma sample pre and post EXO-Prep treatment shows isolation of almost the entire nanoparticle population. The number of nanoparticles isolated by EXO-Prep (8.67x10^11) closely corresponds to the estimated number present in whole plasma (8.92x10^11). Vice versa, the remaining plasma supernatant is almost completely depleted of nanoparticles (0.91x10^11). Isolated vesicles are suitable for multiple dowstream analyses, such as protein profiling via western blotting (Fig 2) or ELISA (Fig 3) or miRNA marker profiling (Fig 4).

1. NTA and exosome detection in whole plasma (A), EXO-Prep isolated exosomes resuspended in 100 ul of PBS 1x (B) and plasma post EXO-Prep treatment (C).

2. Detection of exosome marker Alix in protein lysates (30 μg) from exosomes isolated with EXO-Prep from 100 μl and 500 μl of plasma/serum and 10 and 20 ml of whole urine. Protein lysates (30 μg) from exosomes purified by ultracentrifugation (UC) were used as control.

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Exosome Isolation Tools

EXO-Prep guarantees exosome isolation from complex biofluids, high performances and cheaper price than competitor products EXO-Prep is a fast and efficient method for exosome isolation from biofluids or cell culture media. In particular, when compared to a competitor product on the market, EXO-Prep allows exosome isolation from a smaller volume (100 ul) of complex biofluids as plasma, without thrombin pretreatment or difficult solubilization of exosome pellet. EXO-Prep-isolated exosomes are suitable for different analyses: protein profiling via western blotting (Fig 2) or ELISA (Fig 3), nucleic acids extraction and miRNA/mRNA profiling (Fig 4), FACS.

Characteristics

EXO-Prep

Exosome isolation from cell supernatants

Yes

Yes

Exosome isolation from complex biofluids (plasma, serum, urine)

Yes

Yes, but pellet is hard to solubilize

Thrombin pretreatment

Not required

Necessary

Pellet solubility

Easy

Very hard

Plasma exosome isolation

Minimum sample amount

100 μl

250 μl

Isolated exosome are intact and suitable for ELISA assay

Yes

No suitable for ELISA quantification

Suitable for nucleic acids extraction and analysis

Yes

Yes

Price (for 5 ml of reagent)

●●

●●●

Isolation from 250 ul of human plasma

Isolation from 100 ul of human plasma 12

10 9 8

Ratio to background

Ratio to background

Competitor

7 6 5 4 3 2 1 0

10 8 6 4 2 0

I1 EP

I2 EP

I1 Comp I2 Comp

UC 1

UC 2

I1 EP

I2 EP

I1 Comp I2 Comp

UC 1

UC 2

3. CD9 expression of plasma exosomes from two healthy individuals (100 μl and 250 μl) using EXO-Prep (EP) isolation reagent compared to a competitor reagent (Comp) and exosomes purified by ultracentrifugation (UC).

miRNA Analysis

Total RNA yield 20,00

50 45

22,00

40

24,00

30

CT Values

RNA yield (ng)

35

25 20 15

miR21 miR223

28,00

miR451

30,00

10

32,00

5 0

miR16

26,00

UC

Competitor

EXO-Prep

34,00

UC

Competitor

EXO-Prep

4. RNA extraction and profiling of 4 exosome miRNA from exosomes isolated by EXO-Prep, a Competitor reagent and ultracentrifugation (UC). 100 μl of plasma sample have been used.

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2 Applications •

Multiple profiling of exosomal markers from a single sample or screening of a large number of samples.

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Exosome capure and quantification from human biofluids (plasma, serum, urine, saliva).

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Exosome capture and quantification from human or mouse cell media.

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Titration of purified exosomes.

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Capture and enrichment of specific exosome subpopulations.

Exosome Isolation Tools Immunoplates for Exosome capture and isolation HBM Immunoplates are 96 multiwell plates covalently pre-coated with specific exosome-binding antibodies allowing exosome capture and isolation from different sources (cell supernatant, human plasma, serum, urine and saliva). Covalent coating improves the correct orientation of antibodies maximizing the quantity of immunocaptured exosomes and increasing the binding efficiency of the plate. In addition the coating chemistry reduces the aspecific binding of circulating protein complexes and cell debries, and it helps the enrichment of exosome subpopulations. HBM has developed different types of plates for capturing the overall or enriching specific exosome subpopulations (tumoral, neural, glial, monocytes and platelets). Plates are blocked and stabilized for long term storage.

Advantages •

Ready to use.

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Long term storage (up to 2 years).

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No exosome prepurification required (by ultracentrifugation or other methods).

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Small amount of sample required (100 μl per well).

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Suitable for nucleic acid extraction from exosome captured on the plate.

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Flexibility in designing a multiplexing assay.

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Open platform for customized coating solutions .

Capture of overall Exosomes

Plates are coated with antibodies against exosome surface antigens, present on overell exosome population.

- From human biofluids (plasma, serum, urine, saliva) - From cell media of human or mouse cell lines

Capture of specific Exosome sub-population

Plates are coated with antibodies overexpressed in particular pathological conditions on exosome surface.

- Capture from human plasma - Capture and enrichment of tumor, glial, neuro, platelet derived exosomes

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Exosome Isolation Tools Immunoplates for overall Exosome capture Cat. Code

Type of immunoplate

Package size

Coating antibody

Overall Exosome capture from human plasma and urine HBM-POF-CC/T1 or T5

Transparent

1 or 5 plates

Rabbit

HBM-POF-CC/W1 or W5

White

1 or 5 plates

Rabbit

HBM-POF-CC/B1 or B5

Black

1 or 5 plates

Rabbit

Overall Exosome capture from human serum HBM-POS-CC/T1 or T5

Transparent

1 or 5 plates

Mouse

HBM-POS-CC/W1 or W5

White

1 or 5 plates

Mouse

HBM-POS-CC/B1 or B5

Black

1 or 5 plates

Mouse

Overall Exosome capture from human saliva HBM-POSL-CC/T1 or T5

Transparent

1 or 5 plates

Rabbit

HBM-POSL-CC/W1 or W5

White

1 or 5 plates

Rabbit

HBM-POSL-CC/B1 or B5

Black

1 or 5 plates

Rabbit

HBM-POC-CC/T1 or T5

Transparent

1 or 5 plates

Mouse

HBM-POC-CC/W1 or W5

White

1 or 5 plates

Mouse

HBM-POC-CC/B1 or B5

Black

1 or 5 plates

Mouse

Overall Exosome capture from cell media

Immunoplates for capture of specific Exosome sub-populations Cat. Code

Type of immunoplate

Package size

Different plates are available for colorimetric, luminometric or fuorimetric readings (transparent, white and black, respectively).

Storage conditions Unopened: 2 years, stored at 4°C. Opened: 6 month stored at 4°C.

Amount of material to use It is recommended to load 100 μl of sample per well. Whole human plasma and serum can be loaded for exosome capture.

Using urine and cell media, it is recommended to concentrate the sample 10X, in according to our protocol, before to load the sample on the plate.

Coating antibody

Tumor-derived Exosome capture and enrichment from human plasma HBM-PTF-CC/T1 or T5

Transparent

1 or 5 plates

Rabbit

HBM-PTF-CC/W1 or W5

White

1 or 5 plates

Rabbit

HBM-PTF-CC/B1 or B5

Black

1 or 5 plates

Rabbit

Neural-derived Exosome capture and enrichment from human plasma HBM-PNF-CC/T1 or T5

Transparent

1 or 5 plates

Rabbit

HBM-PNF-CC/W1 or W5

White

1 or 5 plates

Rabbit

HBM-PNF-CC/B1 or B5

Black

1 or 5 plates

Rabbit

Glial-derived Exosome capture and enrichment from human plasma HBM-PGF-CC/T1 or T5

Transparent

1 or 5 plates

Mouse

HBM-PGF-CC/W1 or W5

White

1 or 5 plates

Mouse

HBM-PGF-CC/B1 or B5

Black

1 or 5 plates

Mouse

Monocytes/Platelet-derived Exosome capture and enrichment from human plasma HBM-PPP-CC/T1 or T5

Transparent

1 or 5 plates

Mouse

HBM-PPP-CC/W1 or W5

White

1 or 5 plates

Mouse

HBM-PPP-CC/B1 or B5

Black

1 or 5 plates

Mouse

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Type of Immunoplate

Packaging Immunoplates are individually sealed in an opaque aluminium ziplock bag, compliant to pharmaceutical regulations.

Easy to open and resealable.

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Exosome Isolation Tools Performances

Immunoplates allow exosome protein profiling without pre-purification (by ultracentrifuge, chemical precipitation or microfiltration).

HBM plates are useful tools for immunocapturing exosomes from biofluids or culture media, for protein analyses and protein marker profiling. They allow quantitative and qualitative simultaneous analysis of different protein markers (Fig 5) from the same sample, or expression profiling of a single marker in different samples (Fig 6), without exosome pre-purification via ultracentrifuge or other methods. CD9 expression (Fig 7) of immunocaptured plasma exosomes mimic that of the correspondent ultracentrifuged fraction. No significant cross-reactivity is observed with soluble antigens or other vesicleassociated proteins (Fig 8).

0,7

0,8

0,6

0,7 0,6

O.D. 450 nm

O.D. 450 nm

0,5 0,4 0,3 0,2

0,5 0,4 0,3 0,2

0,1

0,1

0 CD9

CD63

CD81

TSTA3

HSP70

TM9SF4

0

Blank

COLO1

1,2

1.4

1

1.2

UC

0,4

DAUDI

HEK293

Blank

1200 G Pellet 10 000 G Pellet

1

0,8 0,6

RAMOS

6. CD63 profiling on exosomes derived from supernatants of different cell lines

OD at 450nm

O.D. 450 nm

5. Common exosomal biomarkers analysis in a healthy donor’s plasma sample

BLCL21

Unf. Plasma

120 000 G and Filtered Pellet

0.8 0.6 0.4

0,2

0.2 0 1

2

3

4

5

6

7

8

9

0

10

0

Plasma samples

7. Comparison of CD9 detection on purified (ultracentrifuged) plasma exosomes vs unfractionated samples in a set of healthy donor’s plasma

0.5

1

1.5

2 2.5 mL of plasma

3

3.5

4

8. HBM plate is selective in capturing purified exosomes (pellet after centrifugation 120000g) and no other circulating microvesicle (pellet 1200g and 10000g)

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Exosome Isolation Tools

Immunoplates enable enrichment in tumor-derived exosome subpopulation from human plasma Tumor-derived Exosome capture Immunoplates are precoated with a proprietary capturing antibody which allows enrichment and selection of exosomes from tumor origin from human plasma, particularly useful for cancer biomarker profiling (Fig 9). Figure 10 shows that by simply using the Tumor-derived exosome plate it is possible to discriminate healthy donors from cancer patient (indicated by a black arrows).

Immunoplate for Tumor-derived exosome capture

0,5

Immunoplate for overall exosome capture

0,45 0,4

180

200

0,3 Tumor enrichment

0,2 0,15 0,1

180

Normalized data

Overall capture

0,25

Normalized data

160 140 120

0,05 0 Mel Adv

Ova E

Ova Adv

80

HD

9. Enrichment in Tumor-derived exosome in early and late stage melanoma (Mel E; Mel ADV) and ovary carcinoma (Ova E, Ova ADV) using Tumor-derived exosome capture plate vs Overall exosome capture plate (blank subtracted).

1

3

2

4

5

140 120

9

8 6 7 Plate Column

10

11

1

12

2

3

4

5

9

6 7 8 Plate Column

10

11

12

10: Immunoplate for Tumor-derived exosomes capture allows discrimination of cancer patients (black arrows) PLATE2-RAB5

PLATE1-TUCAP1

220 200

180

180

Normalized data

Mel E

160

100

100

Normalized data

O.D. 450 nm

PLATE2-RAB5

PLATE1-TUCAP1

220

0,35

160 140 120

160 140 120 100

100

Specific Immunoplates for enrichment in neural, glial, platelet-derived exosome subpopulation from human plasma 80

0,45 0,4

O.D. 450 nm

0,35 Plate for Neural-derived exosomes

0,3 0,25

Plate for Glial-derived exosomes

0,2 0,15 0,1 0,05 0 SH30

U8730

COLO30

PEP30

11. Specific immunocapture of 30 Îźg of exosome isolated from Neuroblastoma (SH-30) or Glioblastoma (U87-30) cell lines in specific immunoplates. COLO cell and plasma (PEP) purified exosomes were used as controls.

1

2

3

7

6

5 4 Plate Row

Neural-derived exosome capture plate

HD Plasma exosomes in human healthy plasma

0,4

O.D. 450 nm

O.D. 450 nm

0,6

0,2 0 0

5

3

4 5 Plate Row

6

7

8

Glial-derived exosome capture plate

1 SH exosomes in human healthy plasma

2

These plates are precoated with exosome associated antibody indicative of neurological, glial or monocyte/platelet origin enabling specific enrichment of exosomes from human plasma samples. In the examples reported (Fig 11) we show the specificity of binding of exosome derived from neuroblastoma (SH) or glioblastoma (U87) cell lines when plates for neural or glial-derived exosome capture are used. In figure 12 and 13, increasing amount of purified exosomes from neuroblastoma (SH) or glioblastoma (U87) cell lines were spiked in human plasma from healthy donors. Selective capture of specific exosome subpopulation was tested comparing the same plasma sample after spiking of purified plasma exosomes (HD). In both experiments (Fig 12, 13) the signal relative to the specific exosomes subpopulation increases after spiking, while no changes are detectable with HD, suggesting the enrichment of neural or glial derived exosomes respectively.

1,2

0,8

1

8

10

1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

U87 exosomes in human healthy plasma HD Plasma exosomes in human healthy plasma

0

Exosome ug added in human plasma

2

4

6

8

10

Exosome ug added in human plasma

12. Immunocapture of neuroblastoma derived exosomes (SH) subpopulation diluted in human plasma from healthy donors.

13. Immunocapture of glioblastoma derived exosomes (U87) subpopulation diluted in human plasma from healthy donors.

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2 Applications •

Overall exosome isolation from cell culture media and human biofluids (tested for plasma, serum, urine).

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Overall exosome isolation from mouse biofluids (tested for plasma and serum).

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Capture and enrichment of human exosome subpopulation (tumorderived).

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Downstream exosome marker profiling.

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Nucleic acids extraction

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Exosome elution from immunobeads.

Exosome Isolation Tools Latex Immunobeads for Exosome capture and isolation HBM provides several types of Immunobeads for capturing and isolating overall or specific exosome sub-populations. Latex immunobeads are covalently coupled with antibodies against exosome surface antigens, allowing exosome capture from human biofluids (tested for plasma, serum and urine) and cell culture media without pre-purification steps (ultracentrifuge or other method for exosome purification).

HBM immunobeads are able to capture the overall exosome population (Immunobeads for Overall Exosome capture) or to enrich exosome subpopulation derived from tumoral source (Tumoral-derived exosome capture and enrichment). Immunobeads are supplied with an Exosome Elution Buffer, that allows detachment and elution of captured exosomes for downstream analyses, and with a Beads Regeneration Buffer to regenerate immunobeads for further usage. All latex Immunobeads are available in two sizes (0.4 and 1 micron of diameter) and are sold in packages of 10 and 20 reactions, or as TEST format including material for 3 and 5 reactions.

Advantages •

Ready to use.

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Easy, fast and efficient protocol.

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Small sample volume of biofluid or cell culture medium.

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No ultracentrifugation or other methods for exosome purification required.

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Supplied with buffer for exosome elution from beads.

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Immunobeads can be regenerated with Beads Regeneration Buffer and reused.

Beads are mixed together with the sample and incubated over night (ON) for exosomes binding. After ON incubation beads can be easily recovered by centrifugation and washed with appropriate buffer. Bead pellet can be directly used for nucleic acid extraction or protein analysis. Alternatively exosomes can be eluted off the beads with the Exosome Elution Buffer and used for functional studies.

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Exosome Isolation Tools Latex Immunobeads for Exosome capture Cat. Code

Immunobead diameter

Package

Coating antibody

Overall Exosome immucapture from human biofluids HBM-BOLF-CC/10-04

0.4 micron

10 reactions

Mouse

HBM-BOLF-CC/10-1

1 micron

20 reactions

Mouse

HBM-BOLF-CC/20-04

0.4 micron

10 reactions

Mouse

HBM-BOLF-CC/20-1

1 micron

20 reactions

Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions.

Cat Code: HBM-TBOLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm) Overall Exosome immunocapture from cell culture media HBM-BOLC-CC/10-04

0.4 micron

10 reactions

Mouse

HBM-BOLC-CC/10-1

1 micron

20 reactions

Mouse

HBM-BOLC-CC/20-04

0.4 micron

10 reactions

Mouse

HBM-BOLC-CC/20-1

1 micron

20 reactions

Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions.

Cat Code: HBM-TBOLC-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm) Tumor-derived Exosome immunocapture from human biofluids HBM-BTLF-CC/10-04

0.4 micron

10 reactions

Rabbit

HBM-BTLF-CC/10-1

1 micron

20 reactions

Rabbit

HBM-BTLF-CC/20-04

0.4 micron

10 reactions

Rabbit

HBM-BTLF-CC/20-1

1 micron

20 reactions

Rabbit

Immunobeads are also available in TEST format for 3 and 5 reactions.

Cat Code: HBM-TBTLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm) Overall Exosome immunocapture from mouse plasma and serum HBM-BMLF-CC/10-04

0.4 micron

10 reactions

Mouse

HBM-BMLF-CC/10-1

1 micron

20 reactions

Mouse

HBM-BMLF-CC/20-04

0.4 micron

10 reactions

Mouse

HBM-BMLF-CC/20-1

1 micron

20 reactions

Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions.

Cat Code: HBM-TBMLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm) Overall Exosome immunocapture from mouse cell media HBM-BMLC-CC/10-04

0.4 micron

10 reactions

Mouse

HBM-BMLC-CC/10-1

1 micron

20 reactions

Mouse

HBM-BMLC-CC/20-04

0.4 micron

10 reactions

Mouse

HBM-BMLC-CC/20-1

1 micron

20 reactions

Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions.

Cat Code: HBM-TBMLC-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

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Storage conditions Immunobeads must be stored at 4°C.

Amount of material to use Recommended starting volume from 0.1 ml - 0.5 ml of plasma, from 0.5 ml to 1 ml of serum.

Concentrated (10X) urine and cell culture medium samples are recommended prior capture according to our suggested protocol.

Packaging Immunobeads (10 and 20 reaction packages) are supplied with Exosome Elution Buffer, for eluting intact exosomes from beads and with Bead Regeneration Buffer, for regenerating immunobeads that can be reused at least once more.

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Exosome Isolation Tools Performances

Immunobeads are an useful tool for exosome protein profiling Following incubation, beads were recovered by centrifugation, resuspended in Laemmli buffer, and loaded on polyacrylamide gel for western blotting analysis. Exosome were detected with an anti-Alix antibody (human plasma and cell supernatant as described in fig 14 and 15 respectively).

COLO1 Cell Medium

1, 3 and 5: Immunocapture with immunobeads from 1 ml, 0.5 ml and 100 Îźl of human plasma respectively.

2, 4, 6: Ultracentrifuged exosomes from plasma after immunobeads capture.

7 Immunocapture with immunobeads isotype coupled.

8, 9: Ultracentifuged exosome from 0.5 ml and 1 ml of human plasma.

Isotype coupled

HBM-BOLC immunobeads

14. Alix expression by western blotting of human plasma exo- 15. Alix expression by western blotting of exosomes captured on somes captured on HBM-Immunobeads from 0.5 ml of plasma in HBM-BOLC immunobeads from COLO1 cell supernatant vs isocomparison with exosomes purified via ultracentrifuge. 1, 3 and 5: type coupled beads.

Immunocapture with immunobeads from 1 ml, 0.5 ml and 100 Îźl of human plasma respectively. 2, 4, 6: Ultracentrifuged exosomes from plasma after immunobeads capture. 7 Immunocapture with immunobeads isotype coupled. 8, 9: Ultracentifuged exosome from 0.5 ml and 1 ml of human plasma.

Immunobeads enrich for Tumor-derived exosome subpopulation in cancer patients Immunobeads for tumor-derived exosome capture can be used for enriching exosome subpopulations derived from tumoral sources thus providing a novel platform for cancer biomarker research.

Immunobeads for Overall Exosome capture

Immunobeads for Tumor derived exosome capture

16. Anti-Alix WB analysis on exosomes immunocaptured with beads for overall (HBM-BOLF) and for tumor-derived exosomes (HBM-BTLF). WB shows the capture of exosomes only for cancer patients when beads for tumor-derived exosomes are used.

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Exosome Isolation Tools

2

Latex Immunobeads are suitable for exosome RNA extraction and RNA marker profiling Immunobeads allow total RNA extraction from captured exosomes. Isolated total RNA is suitable for mRNA or small RNA analysis. In figure 17 is shown the expression profile of TM9SF4 transcript in cancer cell derived exosomes.

17. Profile of two mRNA marker in exosomes derived from 9 cancer cell lines, previously immunocaptured using HBM beads.

Exosomes can be succesfully eluted from immunobeads which can be reused twice more Exosomes immunocaptured from 1ml of human plasma using latex beads were eluted with Exosome Elution Buffer and then quantified onto HBM immunoplate for overall exosome capture from plasma (Fig 18). Detection was performed using a proprietary anti-CD9-Biotin conjugated antibody. Eluted beads can be regenerated with Bead Regeneration Buffer and reused for capturing exosome two more times (Fig 19).

1,2

O.D.450 nm

1 0,8 0,6 0,4 0,2 0 Unfractioned Plasma

Eluted exosomes

PEP-30

Blank

19. Western Blotting analysis of immunocaptured exosomes on beads.

1- Exosomes immunocaptured with fresh beads 2- Exosomes immunocaptured with beads reused once 3- Exosomes immunocaptured with beads reused twice 4- Exosomes immunocaptured with beads reused the third time

18. CD9 expression in exosomes eluted from immunobeads

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Exosome Isolation Tools

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3

Exosome Quantification Kits

Summary Section 3 Introduction

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ExoTEST

31

TM

Ready to Use ELISA quantification kit

ExoTESTTM performances

33

Exo-FACS Ready to Use kit

35

Exo-FACS performances

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Exosome Quantification Kits Introduction

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Technical support For informations and techincal support contact us at: info@hansabiomed.eu

Exosome research is a relatively novel and exciting field of biomedical research with development of applications in vaccines and in cancer. The use of exosomes for assessment of functional state of parent cell in basic and clinical research is currently hampered by the lack of reliable, affordable and optimized tools for quantitative analysis of exosomes in complex biological samples (e.g. plasma or urine). In this section we present ExoTESTTM an innovative technology that allows purification, quantification and characterization of circulating exosomes specific to a cell type or tissue, or to a condition (health vs. disease). Together with ExoTESTTM HBM team has developed a new kit for FACS (Fluorescence Activated Cell Sorting) analysis of exosome markers, Exo-FACS, which allows exosome FACS profiling without wasting time in exosome isolation by ultracentrifugation.

1- ExoTESTTM Ready to use kit: ELISA assay for exosome immunocapture, quantification and characterization from human biofluids (plasma, serum, urine, saliva) and cell culture media.

2- Exo-FACS Ready to use kit Ready to use kit which allows exosome isolation from biofluids or cell media and biomarker analysis via FACS.

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Exosome Quantification Kits ExoTESTTM Ready to use ELISA Quantification kits ExoTESTTM is a patented double sandwich ELISA assay for quantitative and qualitative analysis of exosomes. In particular ExoTESTTM is a successful platform for exosome quantification and characterization from small amount of human biological fluids or cell media and it may be exploited to identify exosomes released by cancer cells in the plasma and urine of tumor patients in various disease conditions.

Applications •

Exosome capture and quantification from human biofluids and cell culture media.

•

Exosome comprehensive profiling.

•

Pre-clinical research on non-invasive biomarkers for detection and monitoring of a number of pathological conditions (inflammation, cancer, neurodegeneration, etc).

Advantages ExoTESTTM consists in ELISA plates pre-coated with proprietary pan-exosome antibodies enabling specific capture of exosomes from different biological samples, including cell culture supernatants and human biological fluids. Quantification and characterization of exosomal proteins is subsequently performed using appropriate detection antibodies against exosome associated antigens that can be for either generic or cell/tissue-specific exosomes. Lyophilized Exosome Standards, characterized for protein content and particle number (NTA) allow the quantification of unknown sample by a standard calibration curve.

Kit components

Description

Immunoplate

96 well (12 strips x 8 wells) precoated with specific exosome capturing antibody.

Lyophilized Exosome Standards

Exosome Standards from human plasma, serum, urine, saliva or cell medium for calibration curve.

Antibodies for exosome marker detection

Primary anti-human CD9 antibody (HBM proprietary) and secondary antibody HRP conjugated for exosome detection.

Buffers

Sample buffer for antibody dilution and incubation. Washing buffer for washing ELISA plate.

Reagents

Reagents for signal detection.

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•

Ready to use.

•

No initial exosome purification required.

•

User friendly and suitable for multiple marker analyses.

•

Available in TEST format (limited to 3 ELISA strips, 24 wells).

3 Amount of material to use: Starting material: 100 μl of biological sample. Whole plasma and serum can be directly loaded on the plate.

It is recommeded to use concentrate (10X) urine or cell media prior capture according to our suggested protocol.

Lower detection limit: The detection limit of the assay is lower than 0.35 μg of overall exosomes which is the equivalent to less than 50 pg of targeted exosome protein.

Exosome Quantification Kits ExoTESTTM Ready to use ELISA Quantification Kits HBM offers different types of ExoTESTTM kit for quantification of Overall Exosome population from human biofluids (plasma, urine, serum) and from cell culture supernatants. In addition is available the ExoTESTTM for Tumor-derived Exosome Enrichment and quantification. This kit includes an immunoplate able to enrich the tumor exosome subpopulation and is especially suitable for biomarker discovery in clinical cancer research or in development of diagnostics. All ExoTESTTM kits are available with different immunoplate format for colorimetric (transparent) or luminometric (white) or fluorimetric (black) methodology.

Cat. Code

Description

Immunoplate

ExoTESTTM for Overall Exosome immunocapture and quantification from human plasma and urine HBM-RTK-POF/## ExoTEST

TM

HBM-RTK-POS/##

Exosome detection performed with anti-human CD9 antibody (HBM proprietary).

Secondary antibody: anti-mouse HRP conjugated.

Transparent White

for Overall Exosome immunocapture and quantification from human serum Exosome detection performed with anti-human CD9 antibody biotin conjugated (HBM proprietary). Signal detected with Streptavidin-HRP conjugated.

Transparent White

ExoTESTTM for Overall Exosome immunocapture and quantification from cell culture media

Assay calibration: Kit contains Lyophilized Exosome Standards for assay calibration.

Storage conditions: All reagents are shipped and can be stored at 4°C as described in the datasheet of the product.

HBM-RTK-POC/##

Exosome detection performed with anti-human CD9 antibody biotin conjugated (HBM proprietary). Signal detected with Streptavidin-HRP conjugated.

Transparent White

ExoTESTTM for Tumor-derived Exosome immunocapture and quantification from human plasma HBM-RTK-PTF/##

Exosome detection performed with anti-human CD9 antibody (HBM proprietary).

Secondary antibody: anti-mouse HRP conjugated.

Transparent White

Custom made ExoTESTTM for Specific Exosome immunocapture and quantification HBM-RTK-CMK

HansaBioMed offers the flexibility of creating and designing your own kit by choosing among a wide variety of reagents available in our catalog. For information contact info@exotest.eu

All kits are also available in TEST format, limited to 3 ELISA strips (24 wells). Cat Code: HBM-TRTK-###

References

Tetta, Ciro, et al. “Extracellular vesicles as an emerging mechanism of cell-to-cell communication.” Endocrine 44.1 (2013): 11-19.

Zarovni, Natasa, et al. “Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.” Methods (2015).

Vishnubhatla, Indira, et al. “The Development of Stem Cell–Derived Exosomes As a Cell-Free Regenerative Medicine.” J. Circ. Biomark 143 (2014): 2.

Logozzi, Mariantonia, et al. “High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.” PloS one 4.4 (2009): e5219.

32

3

Exosome Quantification Kits Performances

ExoTESTTM presents high sensitivity in detecting low exosome amount, high affinity and low background for exosome binding and quantification.

The sensitivity of the ExoTEST™ was compared to Western blot. The sensitivity of the ExoTEST™ is higher than Western blotting as reported in Figures 1 and 2 showing that 10 μg of lyophilized exosomes are equivalent to 0.1 ng of recombinant exosomal protein. Since the standard curve’s lower concentration is 0.39 μg of lyophilized exosomes (Fig 1), the sensitivity of our test is around 39 pg of protein equivalent. In addition, immunoplates used for exosome capture shows a low background which makes it a reliable, efficient and accurate test (Fig 1).

2 R² = 0,9968

O.D. 450 nm

1,5

1

0,5

0 0

10

20

30

40

50

Lyophilized Exosomes

60

exosome ug

1. CD9 titration (blue line) of plasma healthy donor exosome standards (HBM-PEP100) and comparison with observed background (red line, only secondary antibody).

Recombinant Protein

2. CD9 exosome marker detection by Western Blotting on lyophilized exosomes from human plasma (HBM-PEP100) and recombinant CD9 protein.

ExoTESTTM is a sensitive method for exosome quantification in human biofluids.

ExoTEST enables reliable and precise quantitative measurement and comparison among samples and individual experiments. Standard exosome preparations are provided for designing standard curves and assay calibration. ExoTEST ensures concentration-dependent detection of exosomal markers from different biological samples including human biofluids such as plasma and urine. This assay provides increased sensitivity in detection of exosomal markers with respect to other analytical methods (i.e FACS, WB) We report an example of exosome quantification performed in 5 unknown plasma samples from healthy donors using the ExoTESTTM Ready to use kit for Overall Exosome quantification from human plasma (HBMRTK-POF/TP). Following Lyophilized Standards and unknown samples binding onto the ELISA plate, test is run in according to the kit protocol and exosome detection is performed with anti-CD9 antibody (HBM).

2

0,8 0,7

O.D. 450 nm

1,6

O.D. 450 nm

0,9

y = 0,0349x + 0,0654 R² = 0,9979

1,2 0,8

0,6 0,5 0,4 0,3 0,2

0,4

0,1 0 0

10

20

30

40

50

0

60

#1

Exosome µg

#2

#3

#4

#5

Whole plasma samples

3. Standard curve obtained with Lyophilized Exosome Standards from human plasma healthy donors (HBM-PEP100) with anti-CD9 antibody.

33

4. CD9 titration of exosomes in 5 different whole plasma from healthy donor samples.

3

Exosome Quantification Kits

Exosome quantification is finally performed calculating the quantity of exosomes (expressed in μg) into the 5 unknown samples through the equation of the standard curve (Fig 3). The particles number contained in 100 μl of plasma is calculated from quantity of exosomes (expressed in μg) in according to the particles concentration (number of particles/ml) indicated in the label of the Lyophilized Exosome Standards (HBM-PEP100, NTA: 3x10^11 particles/ml).

Plasma sample

O.D. 450 nm

Exosome μg

Particle number in 100 μl of plasma

#1

0,5673

12,869

3,86x10^9

#2

0,6194

14,205

4,26x10^9

#3

0,4425

9,6692

2,90x10^9

#4

0,3100

6,2717

1,88x10^9

#5

0,7853

18,458

5,54x10^9

34

3

Exosome Quantification Kits Exo-FACS Ready to use kits Exo-FACS allows exosome isolation from biofluids or cell culture media and FACS analysis of exosome markers. The kit consists of EXO-Prep reagent, for exosome isolation, 4 µm beads used for the overall capture of pre-isolated exosomes, lyophilized exosomes from cell culture supernatants or human biological fluids, as positive control. The characterization of exosomal proteins (membrane expressed or internal) is subsequently performed using appropriate detection antibodies against exosome associated antigens.

Applications •

Exosome isolation and exosome marker characterization via FACS.

•

Exosome comprehensive profiling.

Advantages

HBM offers different Exo-FACS kits for staining of exosomal markers from human biofluids (plasma, urine, serum, saliva) and from cell culture supernatants. Exo-FACS contains reagents for 20 reactions (lyophilized exosomes, beads, antibodies and buffers). Primary antibody included in the kit is against a common exosomal marker (CD9 or CD63) and can be used as a positive control for protein profiling via FACS analysis.

Cat. Code

Description

Lyophilized Exosome Standard

Detection antibody

•

Ready to use.

•

No initial exosome purification required.

•

Lyophilized Exosome Standards for positive control included

•

User friendly and suitable for multiple marker analyses.

Kit components •

EXO-Prep, for exosome isolation.

•

Lyophilized Exosome Standards, for positive control.

•

Primary antibody for exosome marker detection as positive control.

•

Secondary antibody Alexa 488.

•

Sample buffer, for antibody incubation.

Exo-FACS ready to use kits for analysis of exosome marker from human biofluids HBM-FACS-PEP

FACS analysis of plasma exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PES

FACS analysis of serum exosomes

HBM-PES100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PEU

FACS analysis of urine exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PESL

FACS analysis of saliva exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

Exo-FACS ready to use kits for analysis of exosome marker from cell culture media HBM-FACS-C

FACS analysis of cell derived exosomes

HBM-###100 * 1 vial, 100 μg

Anti human CD63

* Possibility to choose the Exosome Standard from the list of Lyophilized Exosome Standards from cell media available in the section 1 of this catalog, page 5 and 6.

Storage All reagents are shipped and must be stored a 4°C

35

3

Exosome Quantification Kits Performances

Exo-FACS is an useful tool for exosome protein profiling by using FACS technique Exo-FACS was used for a protein marker profile in exosomes derived from different sources. Exosome binding on FACSbeads was performed by incubation at 4째C over night. Exosome-bead complex is ready to be labeled with fluorophoreconjugated antibodies for specific exosome markers. In figure 5 is shown shown a profile of expression of three different exosome markers in exosomes purified from Melanoma (MM1), Neuroblastoma (SH) and Glioblastoma (U87) cell supernatants.

5. FACS profiling of exosomal markers CD9, CD63 and TM9SF4 in purified exosomes from MM1, SH-SY5Y and U87 cell lines.

36

4

Exosome RNA Extraction Kits

Summary Section 4 Introduction

38

HBM Exosome RNA extraction kits

39

Performances

41

4

Exosome RNA Extraction Kits Introduction

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Exosomes shuttle functional RNA molecules in the target cell. Increasing evidence suggests a role for exosome-derived miRNAs in the development and/or progression of specific human diseases. Pathogenic miRNAs might be exploited as novel therapeutic targets or disease biomarkers in complex diseases, including cancer. In fact, miRNAs seem to play critical roles as transcriptional and post-transcriptional regulators of epigenetic mechanisms and cell processes and have been linked to the etiology, progression and prognosis of cancer. Similar miRNA expression patterns between tumor tissue samples and circulating exosomes have been observed.

HBM has developed optimized solutions for the eďŹƒcient extraction of high-quality total RNA (miRNA and mRNAs) from the overall exosomes and microvesicle population or from tumor-specific exosome subpopulation, which helps to facilitate the identification of tumor miRNA or mRNAs signatures from human biofluids or cell culture media.

Info and Technical support For informations and techincal support contact us at: info@hansabiomed.eu

HBM offers three different kits for exosome capture and RNA extraction.

1- EXO-TotalRNA: Overall Exosome Immunocapture and RNA extraction kit:

Kit allows, by immunocapture, the isolation of overall exosome population from human biofluids or cell culture media and RNA extraction.

2- TumorEXO-TotalRNA: Tumor Exosome Immunocapture and RNA extraction kit: Kit allows, by immunocapture, the enrichment of tumor-specific exosome population from human biofluids and RNA extraction.

3- RNA Basic Kit:

Kit allows RNA extraction from exosomes pre-isolated with different methods (ultracentrifugation, chemical precipitation, immunocapture, size-chromatography etc.)

38

4

Exosome RNA Extraction Kits HBM Exosome RNA extraction kits HBM immunobeads can capture the overall exosome population from a wide range of media, including cell culture supernatants and human biofluids (plasma, serum, urine, etc.) as well as enrich some specific exosome subpopulations (tumor-derived exosomes). The captured exosomes are subsequently lysed with an optimized lysis buffer and total RNA is purified using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80°C. Exosome standards for positive control are also included in the kit.

All our kits guarantee high specificity for exosomal RNA and high yield of total RNA (including small RNAs).

Applications •

Direct capture and exosome RNA extraction from human biofluids and cell culture media without initial exosome purification step.

•

Simultaneous miRNA and mRNA profiling (qRT-PCR, RT-PCR, microarray).

•

Enrichment of tumorderived exosomes for cancer miRNA marker profiling.

Advantages

Cat. Code

Description

Size

EXO-TotalRNA Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media) HBM-RNA-BOF-10/# HBM-RNA-BOF-20/#

RNA extraction from overall exosome population captured with immunobeads

10 reactions 20 reactions

TumorEXO-Total RNA Tumor-derived Exosome Immunocapture and RNA extraction Kit (from human biofluids) HBM-RNA-BTF-10/# HBM-RNA-BTF-20/#

RNA extraction from tumor-derived exosome population captured with immunobeads

10 reactions 20 reactions

RNA Basic Kit* HBM-RNA-B20

RNA extraction from pre-isolated exosome

HBM-RNA-B30

20 reactions 30 reactions

*RNA basic kit does not contain immunobeads for exosome isolation. It is designed for exosome total RNA (miRNA + mRNAs) from pre-isolated exosomes. Compatible with exosome isolated via ultracentrifuge, chemical precipitation, size chromatography, immunocapture etc.

39

•

High yield of total RNA (including small RNAs).

•

Fast and user friendly protocol.

•

Small starting amount of sample (less than 1 ml).

•

Better RNA yield than similar products.

•

The only kit on the market providing Exosome Standards as control.

4 Amount of starting material Whole human plasma and serum can be directly used for RNA extraction. Minimum volume required is 100 μl for plasma and 500 μl for serum loaded for exosome capture.

Using urine and cell media, it is recommended to concentrate the sample 10X, in according to our protocol, before to incubate the samples with immunobeads.

Shipment and storage Kit is shipped a 4°C and all reagents must be stored at 4°C.

Exosome RNA Extraction Kits

Kit components

Description

EXO-TotalRNA Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media) Immunobeads

Immunobeads for overall exosome capture from human biofluids or cell culture media.

Beads Washing buffer (5X)

Buffer for washing Immunobeads after exosome capture.

Lysis buffer

Buffer for exosome lysis.

RNA Washing buffer

Buffer for RNA washing in spin column.

Elution Buffer

Buffer for RNA elution from spin column.

Columns and Tubes

Spin columns for RNA purification and tubes for RNA collection.

Lyophilized Exosome Standard

Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3).

TumorEXO-Total RNA Tumor-derived Exosome Immunocapture and RNA extraction Kit Immunobeads

Immunobeads for tumor-derived exosome capture and enrichment from human biofluids.

Beads Washing buffer (5X)

Buffer for washing Immunobeads after exosome capture.

Lysis buffer

Buffer for exosome lysis.

RNA Washing buffer

Buffer for RNA washing in spin column.

Elution Buffer

Buffer for RNA elution from spin column.

Columns and Tubes

Spin columns for RNA purification and tubes for RNA collection.

Lyophilized Exosome Standard

Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3).

Lysis buffer

Buffer for exosome lysis.

RNA Washing buffer

Buffer for RNA washing in spin column.

Elution Buffer

Buffer for RNA elution from spin column.

Columns and Tubes

Spin columns for RNA purification and tubes for RNA collection.

RNA Basic Kit

40

Exosome RNA Extraction Kits

4

Performances EXO-Total RNA allows extraction of high quality of exosome-derived RNAs from low volumes of sample and better performance than competitors.

Efficiency of EXO-TotalRNA kit was tested vs a competitor kit for RNA extraction from plasma derived exosomes. Extraction of total exosome RNA was performed from 100 μl of healthy donor plasma (HD #1 an #2) with Competitor Kit or with the EXO-TotalRNA Kit (HBM). RNA quality was evaluated by electopherogram (Fig 1) with Small RNA microfluidic chips (Agilent 2100 Bioanalyzer). RNA yield was quantified by Nanodrop (Fig 2) and extracted RNA was subsequently retrotranscribed using the miScript II RT kit (Qiagen). miR-21 and b-actin markers were amplified by qPCR (Fig 3 and 4).

HD#1

HD#2 Competitor Kit

EXO-Total RNA Kit (HBM) 1. Electropherograms of small RNA extracted with HBM EXO-Total RNA kit and Competitor (Agilent 2100 Bioanlyzer)

Total RNA yield (ng) 600 500

PCR amplification miRNA21

25

Ct Values

400

RNA ng

24

26

300 200

27

100 0 HBM

Competitor

2. Nanodrop quantification of total RNA yield

28 HBM

Competitor

3. miRNA 21 amplification by qPCR

41

4. β-Actin amplification by PCR

4

Exosome RNA Extraction Kits

TumorEXO Total RNA allows enrichment of cancer derived miRNA from plasma samples.

Total Exosome derived RNA was extracted from 100 Îźl of plasma from a healthy donor and a cancer patient using RNA kit EXO-Total RNA (HBM-RNA-BOF) for immunocapture of overall exosomes and RNA extraction or TumorEXO Total RNA (HBM-RNA-BTF), for tumor-derived exosome enrichment and RNA extraction, respectively. Total RNA extracted was retrotranscribed using the miScript II RT kit (Qiagen) and the expression level of miR-21 and miR-16 were measured relative to control miR451 by qPCR. Usage of TumorEXO Total RNA shows enrichment in miR-21 and miR-16 in cancer patient (Fig 5 and 6). miR-16

HBM-RNA-BOF

7

HBM-RNA-BTF

Fold Change (normalized to miR-451)

Fold change (normalized to miR-451)

miR-21 6 5 4 3 2 1 0 No Cancer

Cancer

HBM-RNA.BOF

7

HBM-RNA-BTF

6 5 4 3 2 1 0 No Cancer

Cancer

5. Enrichment in miR-21 expression level in cancer when Tumor- 6. Enrichment in miR-16 expression level in cancer when TumorEXO Total RNA kit is used.

EXO Total RNA kit is used.

42

5

Exosome DNA Extraction Kits

Summary Section 5 Introduction

44

EXO-DNA: Isolation of circulating and Exosome-related DNA

45

Performances

41

5

Exosome DNA Extraction Kits Introduction

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Technical support For informations and techincal support contact us at: info@hansabiomed.eu

Together with RNAs, genomic single or double-stranded DNA and mitochondrial DNA have been recently detected in exosomes and microvesicles.

In particular the majority of doublestranded DNA seems to be associated with tumor derived exosomes (Thakur BK et al. Cell research 24.6; 2014) where it represents the entire genome of the cancerous tumor from which exosomes were derived. This discovery corroborates the potential of exosomes, which can be easily obtained from a simple blood test. HBM is the first company to develop a kit for the isolation of circulating and Exosome-associated genomic DNA, EXO-DNA.

EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA

44

5

Exosome DNA Extraction Kits EXO-DNA Isolation of circulating and Exosome-associated DNA EXO-DNA Kit combines the ability of EXO-Prep to isolate exosomes from a wide range of biofluids (plasma, urine, serum etc.) or culture supernatants with a user friendly system of DNA purification. Isolated exosomes are lysed with the appropriate lysis buffer and exosome DNA is purified by spin columns and optimized buffers with a fast turnaround time (approximatively 30 minutes). In addition EXODNA Kit provides lyophilized exosomes to be used as quality controls for exosome capture and DNA extraction.

Applications •

Purification of circulating and exosome-associated DNA.

•

Direct exosome capture and DNA purification from biofluids of cell media without time consuming purification steps.

•

Isolation of genomic exosome-associated DNA

by DNAse treatment.

•

Profiling of exosome associated genomic DNA.

Advantages

* Pellet of exosomes after isolation can be treated with Dnase to eliminate cell free circulating DNA. After Dnase treatment DNA extraction from microvesicle proceeds following the same protocol (see example at page 46).

Cat. Code

Description

HBM-DNA-PS-40/# HBM-DNA-UC-20/# HBM-DNA-UC-40/#

Isolation of circulating and exosome associated DNA from plasma and serum.

20 reactions

Isolation of circulating and exosome associated DNA from urine and cell culture media.

20 reactions

Kit components

Time saving procedure.

•

The only kit on the market providing Exosome Standards as control.

•

Nucleic acids extracted from a small volume amount.

•

Possibility to profile together circulating and exosome-associated genes.

Size

EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA HBM-DNA-PS-20/#

•

40 reactions 40 reactions

Description

EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA EXO-Prep

Reagent for exosome isolation.

Lysis buffer

Buffer for exosome lysis.

Washing buffers

Two washing buffers for DNA purification in spin column.

Elution Buffer

Buffer for RNA elution from spin column.

Columns and Tubes

Spin columns for RNA purification and tubes for RNA collection.

Lyophilized Exosome Standard

Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3).

45

5

Exosome DNA Extraction Kits Performances

Exosome-associate DNA is suitable for point mutation analysis by allele-specific PCR. Healthy donor serum was spiked with 100 Îźg of purified exosome from BRAFV600E-positive A375 melanoma cell lines.

Vesicles were isolated by chemical precipitation with EXO-Prep contained in EXO-DNA kit and treated with or without Dnase 1, to distinguish circulating + Exosome related and only Exosome related DNA. Following digestion, DNA was extracted with EXO-DNA kit and analyzed by bioanalysis and allele-specific qPCR (Fig 1 and 2).

24

Ct Values (Fluorescence)

26 28 BRAF WT

30

BRAF V600E 32 34 36 - DNase

1. Electropherograms of genomic DNA extracted with EXO-DNA Kit with or without Dnase treatment

+ DNase

2. BRAF WT (wild type) and BRAF V600E amplification by qPCR from DNA extracted with EXO-DNA Kit with or without Dnase treatment.

EXO-DNA Kit guarantees high eďŹƒciency isolation of circulating and EV-associated DNA Amplification of beta-actin from exosome-derived DNA. Exosomes were isolated from serum with our without artificial spike (A375-derived exosomes) using EXO-Prep solution and treated (or not) with DNAse I. DNA was extracted with HBM EXO-DNA kit and competitor and beta actin was amplified by qPCR. 25

Ct Values (Fluorescence)

26 27 28 29 30

HBM

31

Competitor

32 33 34 35 Serum

Serum Dnase

Serum A375

Serum A375 DNAse

3. Beta-actin amplification from exosome-derived DNA, extracted with HBM EXO-DNA Kit and a competitor kit for circulating DNA isolation.

46

6

Antibodies for exosome research

Summary Section 6 Introduction

48

Antibodies for Exosome research list

49

Antibody Specifications

50

6

Antibodies for exosome research Introduction

Ordering informations Products can be purchased directly in our on-line shop: www.exotest.eu/online_orders/ US Customers: Info@galenlabsupplies.com Distributors: www.hansabiomed.eu/index. php/distributors

Technical support

Antibodies are an essential tool for basic research, development of diagnostic tests and therapeutics for human disease. Extracellular vesicles (EVs), including exosomes, express antigens with 3D conformations and/or post-translational modifications that often differ from the cellular counterpart. For this reason, most of the antibodies currently available on the market fail to recognize exosome-associated antigens with sufficient sensitivity and specificity. In collaboration with Exosomics Siena Spa, HBM has validated a list of polyclonal and monoclonal antibodies against common (CD63, CD81, ALIX) and disease- specific (cancer and neurodegenerative diseases) exosomal markers.

For informations and techincal support contact us at: info@hansabiomed.eu

48

6

Antibodies for exosome research Antibodies for Exosome Research Cat. Code

Antibody

Type

Applications •

Exosome marker detection in different techniques.

•

Exosome immunocapture.

Applicartions

Monoclonal Antibodies HBM-CD9-###

Anti human CD9

Mouse monoclonal unconjugated

HBM-CD9B-###

Anti human CD9

Mouse monoclonal biotin conjugated

WB,, ELISA, IP

HBM-CD63-###

Anti human CD63

Mouse monoclonal unconjugated

WB, FACS, ELISA, IP, IHC

HBM-CD41-EM1-###

Anti human CD41

Mouse monoclonal unconjugated

FACS, ELISA

HBM-CD81-EM4-###

Anti human CD81

Mouse monoclonal unconjugated

WB, FACS, ELISA, IP, IHC

HBM-CD44-EM1-###

Anti human CD44

Mouse monoclonal unconjugated

WB, ELISA, IP

HBM-CAV1-D4-###

Anti human Caveolin 1

Mouse monoclonal unconjugated

WB, FACS

HBM-ALIX-###

Anti human Alix

Mouse monoclonal unconjugated

WB, FACS, IF

HBM-SF4-###

Anti human TM9SF4

Mouse monoclonal unconjugated

WB, FACS, IP

Shipment and storage

Anti mouse CD9

Mouse monoclonal unconjugated

WB, FACS, ELISA, IP

•

HBM-CD9M-###

WB, FACS, ELISA, IP, IHC

Polyclonal Antibodies HBM-FLOT-### HBM-RAB5-PR1-### HBM-HSP70-SR1-### HBM-SF4-PR2-### HBM-SF4-PR3-### HBM-SF3-PR1-### HBM-TSTA3-PR1-###

Anti human Flotillin

Rabbit polyclonal unconjugated

WB,FACS, ELISA

Anti human RAB5

Rabbit polyclonal unconjugated

WB, FACS, ELISA, IP

Anti human HSP70

Rabbit polyclonal unconjugated

WB, FACS, ELISA

Anti human TM9SF4

Rabbit polyclonal unconjugated

WB, IP

Anti human TM9SF4

Rabbit polyclonal unconjugated

WB, FACS, IP

Anti human TM9SF3

Rabbit polyclonal unconjugated

WB, ELISA, IHC

Anti human TSTA3

Rabbit polyclonal unconjugated

WB, ELISA

Abreviation list: ELISA: Enzyme-Linked Immunosorbent Assay FACS: Fluorescent-Activated Cell Sorting IHC: Immunohistochemistry IP: Immuno precipitation IF: Immunofluorescence WB: Western Blotting

49

Advantages •

All HBM antibodies are tested and validated for exosome antigen reactivity.

•

Available in 2 sizes: 50 μg and 100 μg.

All HBM antibodies are shipped at 4°C. Storage conditions are described in Antibody datasheet.

6

Antibodies for Exosome Research

Anti human CD9 antibody Description

Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24) belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM offers monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specific antigen on both cell lines and extracellular vesicles.

Cat Num/Amount

HBM-CD9-050 (50 μg) HBM-CD9-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS, IP, IHC

References

- Rampini, S., et al. “Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells.” Lab on a Chip 15.16 (2015): 3370-3379.

- Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.” Analytica Chimica Acta (2015).

Western Blotting 1- MM1 (melanoma cell) lysate (20 µg) 2- MM1 cells purified exosomes (20 µg) 3-Plasma healthy donors purified exosomes (20 µg)

FACS CD9 staining of human plasma purified exosomes

ELISA CD9 detection in purified exosomes from human plasma (PEP) and urine (PEU), 30 µg

Anti human CD9 antibody biotin conjugated Description

Anti-CD9 recognizes an human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24) belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM offers two monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specific antigen on both cell lines and extracellular vesicles.

HBM antibodies are compatible with ELISA, WB and flow cytometry applications.

Cat Num/Amount

HBM-CD9B-050 (50 μg) HBM-CD9B-100 (100 μg)

Type

Mouse monoclonal biotin conjugated

Reactivity

Human

Applications

WB, ELISA, IP

Western blotting 1- COLO1 cell lysates (20 µg) 2- CD9 detection in plasma purified exosomes (20 µg)

ELISA CD9 detection in purified exosomes from human plasma (PEP) and urine (PEU), 30 µg

50

Antibodies for Exosome Research

6

Anti human CD63 antibody Description

Anti-CD63 recognizes an extracellular fragment of CD63, a 56 kiloDalton (kDa), type III lysosomal glycoprotein, belonging to the tetraspanin family. CD63 is expressed by granulocytes, platelets, Tcells, monocytes/macrophages and endothelial cells. CD63 protein is a canonical exosome marker and currently used to characterize exosome populations from a variety of body fluids.

Cat Num/Amount

HBM-CD63-050 (50 μg) HBM-CD63-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS, IP, IHC

FACS CD63 staining of human plasma purified exosomes

ELISA CD63 detection in purified exosomes derived from cell supernatants (COLO1-30, MM1-30, BLCL21-30)

Anti human CD41 antibody Description

Integrin alpha chain 2b, also known as CD41, is an heterodimeric integral membrane protein. It undergoes post-translational modifications that result in two polypeptide chains linked by a disulfide bond. CD41 is expressed on platelets and megakaryocytes, but also on early embryonic hematopoietic stem cells and related exosomes.

Cat Num/Amount

HBM-CD41-EM1-050 (50 μg) HBM-CD41-EM1-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

FACS, ELISA

FACS Staining of CD41in human plasma purified exosomes (PEP-30)

ELISA PEP-30: 30 ug of purified exosomes from human healthy donors plasma.

MM1-30: 30 ug of purified exosomes from MM1 cell supernatant COLO1-30: : 30 ug of purified exosomes from COLO1 cell supernatant

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6

Antibodies for Exosome Research

Anti human CD81 antibody Description

CD81 (TAPA1), a member of the tetraspanin family, is virtually expressed on all nucleated cells, but in particular on germinal center B cells. CD81 forms complexes with other tetraspanin proteins, integrins and coreceptors. CD81 is expressed on exosome membranes. HBM anti-CD81 monoclonal antibody is adapt for specific antigen recognition from cell lysates and exosomes using the techniques indicated below.

Cat Num/Amount

HBM-CD81-EM1-050 (50 μg) HBM-CD81-EM1-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS, IP, IHC

References

- Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.” Analytica Chimica Acta (2015).

Western blotting 1- MM1 (melanoma cell) lysate (20 ug) 2- MM1 cells purified exosomes (20 ug) 3-Plasma healthy donors purified exosomes (20 ug)

FACS CD81 staining of COLO1 cell purified exosomes

ELISA CD81 on purified exosomes from urine (PEU), 30 ug

Anti human CD44 antibody Description

CD44, known also as HCAM, is a 742 aminoacids protein involved in lymphocytes activation, homing and hematopoiesis, CD44 is expressed in multiple isoforms. CD44 is highly expressed in cancer tissues and tumoral-derived exosomes, suggesting a role in tumor progression and metastasis.

Cat Num/Amount

HBM-CD44-EM1-050 (50 μg) HBM-CD44-EM1-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, IP

ELISA PEP-30: 30 ug of purified exosomes from human healthy donors plasma.

MM1-30: 30 ug of purified exosomes from MM1 cell supernatant COLO1-30: : 30 ug of purified exosomes from COLO1 cell supernatant

Western blotting 1- 20ug of whole lysate of MM1 cell supernatant purified exosomes 2- 20 ug of purified exosomes from human plasma of healthy donors (PEP)

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Antibodies for Exosome Research

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Anti human Caveolin antibody Description

The lipid raft-associated protein caveolin-1 (CAV1) is the major component of the inner surface of caveolae, small invaginations of the plasma membrane. Caveolin is a transmembrane adaptor molecule that can simultaneously recognize GPI-linked proteins and interact with downstream cytoplasmic signaling molecules. It is highly expressed on exosomes derived from tumor tissue.

Cat Num/Amount

HBM-CAV1-D4-050 (50 μg) HBM-CAV1-D4-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, FACS

Western blotting 1-20 ug of whole lysate of melanoma cell derived exosomes (MM1) 2- 20 ug of whole MM1 cell lysate

FACS Staining of Cav1 in MM1 purified exosomes

Anti human Alix antibody Description

Alix protein, named also ALG2 interacting protein or PDCD6-interacting protein, is a cytoplasmic protein that interacts with apoptosis associated-proteins. Alix plays an active role in exosome biogenesis and it is a useful internal marker for the analysis of exosomal proteins with western blotting.

Cat Num/Amount

HBM-ALIX-SM1-050 (50 μg) HBM-ALIX-SM1-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, FACS, IF

Western blotting 1- MM1 (melanoma cell) lysate (20 ug) 2- MM1 cells purified exosomes (20 ug) 3-Plasma healthy donors purified exosomes (20 ug)

Immunofluorescence Immunofluorescence staining of HeLA cells.

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6

Antibodies for Exosome Research

Anti human TM9SF4 antibody Description

TM9SF4 (TUCAP1) is a newly discovered tumor-associated protein of unknown function that belongs to the Trans-Membrane 9 Superfamily (TM9SF). These proteins are characterized by the presence of a large variable extracellular N-terminal domain followed by nine putative transmembrane domains and a conserved C-terminal domain. TM9SF4 is mainly expressed on exosomes derived from tumor tissue. HBM offers three different monoclonal antibodies that recognize the protein on both exosomes and cell lysates.

Cat Num/Amount

HBM-SF4-PMG1-050 (50 μg) HBM-SF4-PMG1-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Human

Applications

WB, FACS, IP HBM-MM1 (Melanoma cell Exosome Standards)

Western blotting 1- 20 ug of whole lysate of melanoma cell line 2- 20ug of whole lysate of MM1 cell supernatant purified exosomes 3- 20 ug of purified exosomes from human plasma of healthy donors (PEP)

HBM-PEP (Plasma healthy donor Exosome Standards)

FACS Staining of TM9SF4 on MM1 purified exosomes vs human plasma exosomes (PEP) demonstrates the predominant TM9SF4 expression in exosomes derived from tumoral source

Anti mouse CD9 antibody Description

Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24) belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM offers a monoclonal anti-CD9 antibody unconjugated reactive against mouse antigen and able to identify CD9 in mouse cell of biofluids derived exosomes.

Cat Num/Amount

HBM-CD9M-050 (50 μg) HBM-CD9M-100 (100 μg)

Type

Mouse monoclonal unconjugated

Reactivity

Mouse

Applications

WB, ELISA, FACS, IP, IHC

1

2

Western blotting 1- 20ug of whole lysate of B16F10 cell supernatant purified exosomes 2- 20 ug of exosomes isolated from mouse plasma

FACS CD9 staining of B16F10 mouse cell purified exosomes

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Antibodies for Exosome Research

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Anti human Flotillin antibody Description

Flotillin belongs to the band 7.2/stomatin protein family and appears to be strongly expressed in muscle cells and fibroblasts. Flotillin expression is also correlated with Alzheimer development. Flotillin is highly expressed on exosomes and appears to be involved in exosome release mechanism.

It is considered a common marker for exosomes analyses.

Cat Num/Amount

HBM-FLOT-SR1-050 (50 μg) HBM-FLOT-SR1-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS

References

Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.” Analytica Chimica Acta (2015).

FACS Purified exosomes from MM1 (30 ug) stained by anti-Flotillin antibody

ELISA Detection of different exosomes purified from cell culture supernatants or human plasma (HD), performed with anti-Flotillin

Anti human Rab5 antibody Description

Rab5 is a small GTPase belonging to Ras superfamily of monomeric G proteins. Rab GTPases play an essential role in the regulation of membrane traffic and are involved in vesicle formation and trasport and fusion to the membrane. Rab5 is expressed on exosome membranes and it might have an active role during endo/exocytotic processes of microvesiscles through the plasma membrane.

Cat Num/Amount

HBM-RAB5-PR1-050 (50 μg) HBM-RAB5-PR1-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS, IP

References

Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.” Analytica Chimica Acta (2015).

0.5 ml

0,25 ml

0,1 ml

Western blotting Exosomes purified from 0.5, 0.25, 0.1 ml of human plasma demonstrate different intensity of Rab5 expression

FACS Purified exosomes from COLO1 cell line detected by Rab5 antibody

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6

Antibodies for Exosome Research

Anti human HSP70 antibody Description

Heat shock protein 70 (HSP70) is a molecular chaperone that facilitates the assembly of multiprotein complexes and trafficking of polypeptides across cell membranes. HSP70 is active in promoting tumorigenesis and functions as an anti-apoptotic factor. It is highly expressed on exosomes derived from tumor tissues.

Cat Num/Amount

HBM-HSP70-SR1-050 (50 μg) HBM-HSP70-SR1-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

WB, ELISA, FACS, IP

References

Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform.” Analytica Chimica Acta (2015).

Western blotting Analysis of HSP70 expression in different exosomes derived from tumoral cell lines: 1- COLO1; 2- SH-SYSY; 3 - U87; 4 - SK

FACS Staining of COLO1 derived exosomes with anti-HSP70

Anti human TM9SF3 antibody Description

TM9SF3 belongs to nonaspanins family proteins (TM9SF), characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. Although little is known about the functions of this protein, it might participate in tumor invasion and it is emerging as a potential prognostic factor. Protein is expressed on plasma membranes and exosomes.

Cat Num/Amount

HBM-SF3-PR1-050 (50 μg) HBM-SF3-PR1-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

WB, ELISA,IHC

Western blotting Detection of TM9SF3 in 1 - MM1 cell lines (20 µg) 2 - MM1 derived exosomes (20 µg)

Elisa TM9SF3 detected in different purified exosomes immunocaptured on EXOtest plate

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Antibodies for Exosome Research

6

Anti human TM9SF4 antibody Description

TM9SF4 (TUCAP1) is a newly discovered tumor associated protein that belongs to the TransMembrane 9 Superfamily (TM9SF). These proteins are characterized by the presence of a large variable extracellular N-terminal domain followed by nine putative transmembrane domains and a conserved C- terminal domain. TM9SF4 is mainly expressed on exosomes derived from tumor tissue. HBM offers two rabbit polyclonal antibodies that recognize the protein from both exosomes and cell lysates.

Cat Num/Amount

HBM-SF4-PR2-050 (50 μg) HBM-SF4-PR2-100 (100 μg) HBM-SF4-PR3-050 (50 μg) HBM-SF4-PR3-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

HBM-SF4-PR2: IP, FACS, IHC HBM-SF4-PR3: WB, IP

Western blotting HBM-SF4-PR2 20 ug of whole HEK293 cell lysate (1), 20 ug of exosomes purified from MM1 cell supernatant (2) and from human plasma of healthy donors (3)

FACS Purified exosomes from MM1 cell line detected by HBMSF4-PR1 antibody

IHC Immunohystochemical staining of TM9SF4 in specimens derived from healthy colon.

Anti human TSTA3 antibody Description

Tissue specific transplantation antigen P35B is a NADP(H)-binding protein. It catalyzes the twostep epimerase and the reductase reactions in GDP-D-mannose metabolism, converting GDP4-keto-6-D-deoxymannose to GDP-L-fucose. TSTA3 have been identified in the overall exosome population.

Cat Num/Amount

HBM-TSTA3-PR1-050 (50 μg) HBM-TSTA3-PR1-100 (100 μg)

Type

Rabbit polyclonal unconjugated

Reactivity

Human

Applications

WB, ELISA

Western blotting Detection of TSTA3 in: 20 ug COLO1 purified exosmes.

20 ug of exosomes purified human plasma (PEP) *in purified exosomes from plasma TSTA3 can appear in dimer form (signal around 70 kDa)

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6

Antibodies for Exosome Research

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7

Custom Services and Collaborations

Summary Section 7 Introduction

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Exosome and Microvesicle purification and analysis

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RNA and DNA profiling and Next-Generation-Sequencing

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Custom products

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Additional services and project collaboration

7

Custom Services and Collaborations Introduction

Info and Technical support For informations and techincal support contact us at: info@hansabiomed.eu

Being the oldest company entirely dedicated to exosome research, since 2007 HansaBioMed has developed unique competences and tools allowing us to help you with your research work. Our experienced scientists will help you accelerate your research and achieve your objectives quickly, providing consultancy and/or professional services using the state of art equipment. Our services include:

1- Exosome and Microvesicle purification from different biological samples and complex matrices.

2- Exosome RNA and DNA profiling and Next Generation Sequencing.

3- Custom products (immunoplates for exosome capture, immunobeads, ExoTESTTMkits).

4- Modification of biomolecules.

5- Development of new tools for capture and enrichment of exosome subpopulations or quantification of specific markers.

6- Project collaborations and technical consulting.

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7

Custom Service and Collaborations Exosome and Microvesicle purification and analysis Our Exosome Custom Service offers purification of Exosomes and Microvesicles from a wide range of biological samples (human and animal) and cell culture media. Exosome purification can be obtained using different methods according to your sample characteristics and your requests (combination of ultracentrifugation and microfiltration steps, immunocapture, chemical precipitation). Exosome can be subsequently quantified for total protein content and validated by NTA (NanoSight LM10). Purified exosomes can be shipped to the customer lyophilized or frozen, as required. Moreover, we offer additional services to facilitate your research such as those listed below.

Service

Description

Exosome and Microvesicle Isolation

Exosome and Microvesicle isolation via ultracentrifuge or other methods.

Isolation of exosome miRNA

Extraction of miRNA from exosomes. miRNA will be shipped to the customer in dry ice.

Exosome protein marker profiling

Profiling of exosome protein marker via quantitative ELISA, FACS analysis or Mass Spectrometry.

RNA and DNA profiling and Next-Gen- Sequencing (NGS) Hansabiomed scientists can provide an expert service for isolation and identification of exosome-associated RNA/DNA biomarkers leveraging the throughput and scalability of Ion Torrent and SOLiD System (Thermo Fisher Scientific) next-generation sequencing platforms. This service is an easy solution for researchers who are interested in identifying new exosome RNA biomarkers or in the understanding the profiling expression of such biomarkers in the exosomes of their model cellular systems or patient biofluids. You will receive your data back in approximately 4-6 weeks.

Service

Description

RNA and DNA Next Gen Sequencing

Extraction of RNAs and DNA vesicle associated and NGS service.

Isolation of exosome RNAs or genomic DNA and profiling

Extraction of RNAs (miRNA and/or total RNA) and genomic DNA from exosomes. Profiling of nucleic acid marker by qRT-PCR. Alternatively RNA or DNA can be shipped to the customer in dry ice.

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Advantages •

Exosome purification and services performed by experienced scientists and qualified personnel.

•

Material is processed quickly.

•

HansaBioMed has all the necessary facilities for exosome purification, protein and nucleic acid profiling.

7

Custom Services and Collaborations Custom products HansaBioMed can design and create a personalized kit by choosing from among a wide variety of reagents and tools in our catalog.

Custom product

Description

Custom ELISA immunoplate

1- Choose your preferred plate (trasparent, white or black) depending on your final application.

2- Choose the antibody from our antibody list (see section 6) or send us the antibody for plate coating.

Custom ExoTESTTM Ready to use kits

1- Select the plate/beads that provides the capture or enrichment of exosome population of interest.

2- Select the Exosome Standards you need.

3- Select the primary antibody you want to use for exosome detection.

Custom immunobeads

Latex immunobeads will be covalently coupled with the antibody of your interest, chosen from our antibody list.

For other Custom made solutions and for informations contact us at: info@hansabiomed.eu

Additional services and project collaboration HansaBioMed is open for collaboration with private or academic partners in research activites related to different fields of exosome research and applications.

Contact us for: Service

Description

Biomolecule labeling and modification

We can provide biomolecule modifications such as: protein labeling with fluorescence dyes, conjugation of biomolecules with affinity ligands, PEGyation of biomolecules, immobilization of biomolecules on different surfaces (hydrogels, molded polymers, synthetic membranes) etc.

Development of new assays for exosome marker identification

We can develop personalized ELISA assays for a specific exosome protein marker quantification or qPCR assays for identification of miRNA and DNA markers.

Development of tools for capture and enrichment of specific exosome subpopulations

We can develop tools for capture and isoaltion of specific exosome subpopulations to improve exosome comprehensive profiling in diseases.

Project and collaborations and technical consulting

Contact us for: OEM product development, cell free regenerative medicine, collaboration in research projects (Horizon 2020, Eurostars, NIH grants, etc).

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Cell Fractionation Kits Separe different cell compartments with HansaBioMed Cell Fractionation Kits.

HansaBioMed offers a comprehensive selection of systems for convenient fractionation of different cellular compartments. These kits provide unique formulations of buffers and reagents for convenient preparation of highly enriched cellular fractions from mammalian cells and tissues. The prepared protein fractions are suitable for downstream assays, such as two-dimentional gel, protein immunoblotting, gel-shift assay, enzyme activity assays, reporter assays, and more.

Cell Cytosol fraction

Cytosol extraction

Cell Organells Membrane fraction

Membrane extraction

Nuclei Nuclear fraction

Nuclear extraction

Cytoskelatal fraction

Product code

Product name

Description

HBM-K270-50

Fraction-PREP Cell Fractionation Kit

The Fraction-PREPTM Cell Fractionation System is designed to fractionate cellular proteins into four distinct protein fractions: Cytosolic fraction, membrane fraction, nuclear fraction, and cytoskeletal fraction. All four protein fractions can be directly used in downstream assays, such as 2-D gel, Western blot, gel-shift assay, translocation, enzyme activity assays, and reporter assays, etc.

HBM-K256-100

Mitochondria/Cytosol Fractionation Kit

Separate highly enriched mitochondria from cytosol of mammalian cells.

Simple procedure with no ultracentrifugations or toxic chemicals involved.

HBM-K266-100

Nuclear/Cytosol Fractionation Kit

Separate nucleus from cytoplasm in less than 90 minutes. The kit provides a system that maintains the nuclear and cytosol compartments separated. The separated fractions are suitable for downstream assays.

HBM-K268-50

Membrane Protein Extraction Kit

Isolate integral membrane and/or plasma membrane proteins efficiently from cultured mammalian cells. The isolated membrane proteins can directly be used in downstream assays.

HBM-K269-500

Mammalian Cell Extraction Kit

Extract mammalian proteins from tissues and cells under nondenaturing conditions. Cell extracts can be used in a variety of assays, such as enzyme activity assays, Western blot, and others.

HBM-K280-50

Mitochondrial DNA Isolation Kit

The kit provides unique formulations of buffers and reagents for isolating a highly enriched mitochondrial fraction. Mitochondrial DNA are then isolated using reagents included in the kit

PEG virus Precipitation Kit

HansaBioMed’s PEG Virus Precipitation Kit provides an easy, convenient and time-saving method to concentrate virus without ultra-centrifugation. The kit is able to concentrate retroviruses, baculoviruses, lentiviruses, and phages etc. in cell culture medium or environmental samples.

HBM-K904-###

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Cell Proliferation and Viability Kits Easy and fast analysis of cell viability and cell proliferation assays.

Proliferation or viability analyses are crucial for cell growth and differentiation studies, and are often coupled with metabolism analysis. Metabolic activity is commonly used as a viability indicator, but for some applications it can be important to assess viability independent of metabolic state and this is where the cell viability assays come in play.

Proliferation is also a convenient measure of population dynamics in studies of cytokines or growth factors, or in bioprocess optimization work. HansaBioMed offers a panel of different solutions for a fast and easy determination of the cell viability and proliferation in your cell cultures.

Product code

Product name

Description

HBM-K306-###

BrdU Cell Proliferation Assay Kit

Compared to other cell proliferation assays, this kit detects only the proliferating cells and not the seeded cells. This highly sensitive, non-radioactive kit detects as less as 50-100 proliferating cells.

HBM-K501-100

Live-Dead Cell Staining Kit

The Live-Dead Cell Staining Kit provides the ready-to-use reagents for convenient discrimination between live and dead cells. Stained live and dead cells can be visualized by fluorescence microscopy using a band-pass filter (detects FITC and rhodamine)

HBM-K300-###

MTS Cell Proliferation Colorimetric Assay Kit

MTS Cell Proliferation Assay Kit is a colorimetric method for sensitive quantification of viable cells in proliferation and cytotoxicity assay. . It can also be used for the analysis of cytotoxic compounds like anticancer drugs and many other toxic agents and pharmaceutical compounds

Proteasome Activity Assay Kit

Proteasome Activity Assay takes advantage of the chymotrypsin-like activity, utilizing an AMC-tagged peptide substrate which releases free, highly fluorescent AMC in the presence of proteolytic activity. it allows differentiation of proteasome activity from other protease activity which may be present in samples

HBM-K302-###

Quick Cell Proliferation Assay Kit

The assay is based on the cleavage of the tetrazolium salt to formazan by cellular mitochondrial dehydrogenase. The amount of the dye generated by activity of dehydrogenase is directly proportional to the number of living cells.

HBM-K320-50

Senescence Detection Kit

The Senescence detection kit is designed to histochemically detect SA-尾Gal activity in cultured cells and tissue sections, a known characteristic of senescent cells.

HBM-K771-100

Trypsin Activity Assay Kit

The kit detects 10-100 mU (p-NA unit) trypsin in various samples. . In the assay, trypsin cleaves a substrate to generate p-nitroaniline (p-NA) which is detected at 位=405 nm.

HBM-K304-2500

WST-1 Cell Proliferation Reagent

The ready-to-use cell proliferation reagent, WST-1 provides a simple and accurate method to measure cell proliferation, which is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases.

HBM-K245-100

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HansaBioMed LLC

Contact in US

Contact in Russia and CIS countries

Email: info@hansabiomed.eu Tel: +372 6204348

Email: info@galenlabsupplies.com Tel: 860-704-9058 Fax: 888-260-6091

Email: mail@algimed.by Tel: +375 172 024 302 Fax: +375 172 024 338

Akadeemia tee 15, 12618 Tallinn, ESTONIA www.hansabiomed.eu

Galen Laboratory Supplies P.O. Box 682 Middletown, CT 06457 www.galenlabsupplies.com

Algimed LTD Logoyskiy Tract 22/1-35B 220090 Minsk, BELARUS www.algimed.by